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From HOWDUNNIT: FORENSICS
TOXICOLOGICAL TESTING PROCEDURES
The biggest problem facing the toxicologist is that there are literally thousands of drugs and chemicals that are harmful, addictive, or lethal if ingested, injected, or inhaled. Some even absorb directly through the skin. Toxicological testing is time-consuming and expensive, and few, if any, labs can afford to perform such testing on every case. For this reason, the testing must be as focused as possible.
An understanding of the circumstances surrounding the death is important since clues at the scene often point toward a particular drug. For example, a young girl found on her bed at home with an empty pill bottle at her side would lead to one avenue of testing while a long-term addict found in an alley with fresh needle marks would follow another path. The more clues as to the likely toxin that the circumstances of the death can supply, the narrower the field of possibilities the toxicologist must consider.
THE TWO-TIERED SYSTEM
When testing for drugs or poisons, the toxicologist typically follows a two-tiered approach. Initial tests, called presumptive tests, are for screening purposes and are typically easier and cheaper to perform. When negative, they indicate that the drug or class of drugs in question is not present and further testing is unnecessary. When positive, they indicate that a particular substance possibly is present. By using these screening tests the number of possibilities can be greatly reduced and the toxicologist can move on to the second phase, which utilizes more focused confirmatory testing. These tests are more expensive and time-consuming but are designed to establish the identity of the exact drug present. This two-tiered approach saves considerable time and money.
This same approach is used whether the toxicologist is asked to analyze blood, urine, and other materials obtained from a person (living or deceased) or to test a batch of seized material believed to be illicit drugs.
Let’s say a corpse is found in an alleyway known for methamphetamine sales and use. If blood samples obtained at autopsy show a positive presumptive test for amphetamines, further confirmatory testing to identify the exact amphetamine present is indicated. If the test is negative, no further testing for amphetamines is done and the toxicologist will search for other classes of drugs.
To be doubly certain, the toxicologist prefers to find the drug or poison in at least two separate locations. Finding the toxin in the blood and the liver tissue is more reassuring than finding it in either one alone.
Or let’s say that the toxicologist is asked to test a seized substance and doing so shows a positive presumptive test for cocaine. Further confirmatory testing would then be indicated. If the screening test is negative, the substance may be analyzed for other drugs, but cocaine has been ruled out.
In most labs, testing for controlled and illegal drugs consumes 75 percent of the lab’s time and resources. The areas most often tested in this type of examination are blood and urine. After one of the presumptive tests shows that a particular drug or class of drugs is likely present, confirmatory testing with the combination of gas chromatography and mass spectrometry (GC-MS) or infrared spectroscopy are used to accurately identify which substance is present. See the appendix for details on these procedures.
Presumptive testing comes in many varieties. Common toxicological screening tests are color tests, immunoassays, thin layer chromatography, and ultraviolet spectroscopy.
Tests in which a reagent (any active chemical solution) is added to blood, urine, or tissue extractions, and if the particular chemical tested for is present, a color change reaction will occur. The color change results from a chemical reaction between the drug and the reagent, which produces a new compound that imparts a specific color to the mixture. These tests are cheap, easy, and quick, and can determine if a specific chemical or class of chemicals are present in the material tested. If it does not indicate that the toxin is present, further testing is not necessary.
There are a wide variety of color tests that reveal the presence of many types of drugs. Some of the most common are:
TRINDER’S TEST: This reagent, containing ferric nitrate and mercuric chloride, turns violet in the presence of salicylates (aspirin and similar compounds).
MARQUIS TEST: This reagent contains formaldehyde and sulfuric acid and turns purple in the presence of morphine, heroin, and most opiates, and brownish orange if mixed with amphetamines or methamphetamines.
VAN URK TEST: This is a test for LSD and other hallucinogenic drugs. The reagent is a mixture of dimethylaminobenzaldehyde, hydrochloric acid (HCl), and ethanol. It turns purple to indicate a positive reaction.
DILLIE-KOPPANYI TEST: In this test, the sample is treated with cobalt acetate in methanol and then with isopropylamine in methanol. It turns violet-blue if barbiturates are present.
DUQUENOIS-LEVINE TEST: This three-step test determines if marijuana or other cannabinoids are present. The sample is treated with a mixture of vanillin and acetaldehyde in ethanol, then with HCl, and finally with chloroform. A deep purple color is a positive result.
SCOTT TEST: This is also a three-step test that uses a mixture of cobalt thiocyanate and glycerine, followed by HCl, and then chloroform. Cocaine turns blue after the thiocyanate is added, changes to pink with the HCl, and then blue once again when chloroform is added.
Other Screening Tests
IMMUNOASSAY: Immunoassays, which measure the concentration of a drug in a liquid (see the appendix), are easy, very sensitive, and useful for rapidly screening urine samples for certain drugs. However, the manufactured antibodies can also react with compounds that are very similar to the sought-after drug, a lack of specificity that makes this a presumptive test rather than a confirmatory one.
THIN LAYER CHROMATOGRAPHY (TLC): TLC (see the appendix) not only tentatively identifies many chemicals, but is also useful for separating the components of a sample. Once TLC has tentatively identified a substance, its identity is confirmed with mass spectrometry.
GAS CHROMATOGRAPHY (GC): As with TLC, GC’s (see the appendix) primary use is in making a presumptive identification and separating various compounds from one another. A positive result is confirmed by using mass spectrometry.
ULTRAVIOLET (UV) SPECTROSCOPY: This test takes advantage of the fact that different chemicals absorb UV light in varying amounts (see the appendix). Since it can’t identify the exact compound, it is only useful for screening
A Typical Screening Protocol
Each lab has its own protocol for drug screening. What tests are used and in what order they are performed depends on the available staff and equipment, budgetary restrictions, and the bias of the toxicologist in charge. But most labs have certain standard screens they employ when first confronted with an unknown sample. These basic screens might include:
ALCOHOL SCREEN: GC is used to isolate and identify the various alcohols and related compounds such as acetone.
ACID SCREEN: Immunoassay of urine samples is used to detect acidic compounds such as barbiturates and aspirin.
ALKALI SCREEN: GC screens for substances that dissolve in alkaline solutions. These substances include many tranquilizers, synthetic narcotics, and antidepressants.
NARCOTIC SCREEN: Urine immunoassay reveals opiates, cocaine, and methadone.
By using these general screening procedures, the toxicologist can quickly exclude many commonly encountered drugs and narrow his area of search for those that are present. Based on these results, further screening and confirmatory tests are used to ultimately identify any unknown substance.
A good confirmatory test must possess sensitivity and specificity in that it must recognize the chemical in question (sensitivity) and be able to identify it to the exclusion of all others (specificity). This means that once a chemical has undergone a screening test and a presumptive identity has been established, a confirmatory test will accurately determine the true identity of the unknown substance.
The most important confirmatory test used by the toxicologist is mass spectrometry (MS) (see the appendix). In MS, the sample is bombarded with electrons, which fragment the chemical into ionic fractions. This fragmentation pattern is called a mass spectrum. It is different for each element and compound. This means that it gives a chemical fingerprint of the chemical being tested and can identify virtually any compound. When the mass spectrum of an unknown substance is compared to known reference standards, the identity of the unknown sample comes to light. The National Institute of Standards and Technology (NIST) maintains a database of the mass spectra of known chemicals.
In the forensic toxicology laboratory, MS is usually employed in combination with gas chromatography (GC). This combination is called gas chromatography/mass spectrometry (GC/MS). In GC/MS, gas chromatography is used to separate the test sample into components and MS is employed to identify each component. The GC/MS is as close to being foolproof as any technique available.
Though used less often than MS, infrared spectroscopy (IR) can also determine the chemical fingerprint of the tested substance (see the appendix). Instead of electrons, the substance is exposed to infrared light. When any light strikes an object or substance, it is transmitted (passed through), absorbed, or reflected. When exposed to infrared light, each compound transmits and absorbs the light in its own unique pattern. These unique patterns determine which compounds are present, and thus identify the chemical substance tested. This test is also used in conjunction with GC. This combination is termed GC/IR.
INTERPRETING THE RESULTS
After testing has revealed the presence and concentration of a chemical substance, the hard part begins. The toxicologist must now assess what the results mean. He evaluates each of the drugs present with an eye toward the route the drug was administered and whether the concentrations played a role in the subject’s behavior or death.
Route of Entry
The route of entry of the toxin is very important since it might provide a clue as to whether the victim self-administered the drug or someone else administered the drug. For example, if a drug was injected and the victim possessed no means to do so or if the injection site was in an area that made self-administration unlikely, homicide might be a stronger consideration.
Another important fact is that the concentration of the toxin is usually greatest at the administration site. Ingested toxins are more likely to be found in the stomach, intestines, or liver, while inhaled gases will be concentrated in the lungs. If injected, the drug can often be isolated from the tissues around the injection site. Drugs taken intravenously bypass the stomach and liver, directly enter the bloodstream, and are quickly distributed throughout the body. In this circumstance, the toxicologist may find high concentrations of the drug in the blood and in multiple tissues of the body, but little or none in the stomach and liver as would be seen with ingestion. This will help him determine the route of intake.
Drug Blood Level
Earlier we discussed the concept of bioavailability and how the level of a drug in the blood closely correlates with the drug’s actions and toxicity. This means that finding a large amount of a toxin in the victim’s stomach does not necessarily mean that the drug was the cause of death. The important fact is that drugs in the stomach will not kill. They must first be absorbed into the blood and distributed to the body.
For example, if the toxicologist found a large amount of a tranquilizers in a victim’s stomach, particularly if most of the pills were intact and had not been digested, and also found a low blood concentration of the drug, he would likely conclude that the pills were taken shortly before death and played little or no role in the victim’s demise.
There are exceptions. In cases of caustic acid and alkali (lye or caustic soda) ingestion, the blood levels are not important since these chemicals cause direct contact damage and do not need to be absorbed into the body to do harm (discussed later in this chapter).
Still, in most situations, blood levels are important because they correlate more strongly with the effects of the chemical in question. When the toxicologist determines a blood level of a certain chemical, he might assign it to one of four broad categories:
NORMAL: This would be the level expected in the general population under normal circumstances. An example would be low levels of cyanide. Even though this is a deadly poison, it is found in the environment, and therefore most people have low normal levels of cyanide in their blood. Smokers have even higher levels, but this would still be considered normal.
THERAPEUTIC: This is the level that your doctor strives for. If he gives you an antibiotic or a medication for high blood pressure, he wants to accomplish a blood level of the drug that will bring about a therapeutic effect. Patients with certain cardiac problems may be placed on digitalis. The doctor will periodically draw a blood test to check the therapeutic level of the drug. The reason he does this is that too little will offer less benefit to the patient and too much can cause severe problems since digitalis is potentially a deadly poison.
TOXIC: A toxic level is one that may cause harm or death. When a prescribed drug passes the therapeutic level and reaches the toxic level it has moved from being a medication to being a poison. Using the example of digitalis, a toxic level might lead to nausea, vomiting, and a yellowish tinge to the person’s vision. Or it may cause a deadly change in the rhythm of the heart. These would be toxic effects.
LETHAL: This is the level at which the drug in question would consistently cause death. In toxicology we use the term LD50 to measure a chemical’s lethal potential. The LD50 of a drug is the blood concentration at which 50 percent of people would die.
From this, you might assume that the toxicologist simply has to determine the blood level of any toxin and then he can determine if the level was toxic or lethal. Though that may seem logical, it is far from the truth.
Each person reacts to chemicals and toxins differently. Much of this variance can be related to age, sex, body size and weight, genetics, and nutritional and health status. An individual who is young, robust, and healthy should tolerate more of a given drug than would someone who was old, thin, and sickly. And in general, that is true. As mentioned earlier, a person’s habits also affect how he will react. The toxicologist must consider these facts when assessing whether a given level of a drug is toxic or lethal, or whether it contributed to the subject’s behavior or death.
Acute vs. Chronic Poisoning
At times the toxicologist is asked to determine whether a poisoning is acute or chronic. A good example is arsenic, which can kill if given in a single large dose or if given in repeated smaller doses over weeks or months. In either case, the blood level could be high. But the determination of whether the poisoning was acute or chronic may be extremely important. If acute, the suspect list may belong. If chronic, the suspect list would include only those who had long-term contact with the victim, such as a family member, a caretaker, or a family cook.
So, how does the toxicologist make this determination?
In acute arsenic poisoning, the ME would expect to find high levels of arsenic in the stomach and the blood, as well as evidence of corrosion and bleeding in the stomach and intestines, as these are commonly seen in acute arsenic ingestion. If he found little or no arsenic in the stomach and no evidence of acute injury in the gastrointestinal (GI) tract, but high arsenic levels in the blood and tissues, he might suspect that the poisoning was chronic in nature. Here, an analysis of the victim’s hair can be invaluable.
Hair analysis for arsenic (and several other toxins) can reveal exposure to arsenic and also give a timeline of the exposure. The reason this is possible is that arsenic is deposited in the cells of the hair follicles in proportion to the blood level of the arsenic at the time the cell was produced.
In hair growth, the cells of the hair’s follicle undergo change, lose their nuclei, and are incorporated into the growing hair shaft. New follicular cells are produced to replace them and this cycle continues throughout life. Follicular cells produced while the blood levels of arsenic are high contain the poison, and as they are incorporated into the hair shaft the arsenic is, too. On the other hand, any follicular cells that appeared while the arsenic levels were low contain little or no arsenic.
In general, hair grows about a half-inch per month. This means that the toxicologist can cut the hair into short segments, measure the arsenic level in each, and reveal a timeline for arsenic exposure in the victim.
Let’s suppose that a wife, who prepares all the family meals, slowly poisoned her husband with arsenic. She began by adding small amounts of the poison to his food in February and continued until his death in July. In May he was hospitalized with gastrointestinal complaints such as nausea, vomiting, and weight loss (all symptoms of arsenic poisoning). No diagnosis was made, but since he was doing better after ten days in the hospital, he was sent home. Such a circumstance is not unusual since these types of gastrointestinal symptoms are common and arsenic poisoning is rare. Physicians rarely think of it and test for it. After returning home, the unfortunate husband once again fell ill and finally died.
As part of the autopsy procedure, the toxicologist might test the victim’s hair for toxins, and if he did, he would find the arsenic. He could then section and test the hair to determine the arsenic level essentially month by month. If the victim’s hair was three inches long, the half-inch closest to the scalp would represent July, the next half inch June, the next May, and so on until the last half inch would reflect his exposure to arsenic in February, the month his poisoning began. Arsenic levels are ex-
pressed in parts per million (ppm).
The toxicologist would look at this timeline of exposure and likely determine that the exposure occurred in the victim’s home. The police would then have a few questions for the wife and would likely obtain a search warrant to look for arsenic within the home.
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